7. Project Summary Broad, long-term objectives: The research objectives stated herein, and in future studies that would build upon these findings, seek to reveal general scientific principles in the mechanisms that drive spillover and adaption of zoonotic RNA viruses. Significance: RNA viruses contribute to more than half of all human infectious diseases that have originated as zoonoses. Wildlife studies to form a complete understanding of RNA viral- host interactions, especially those that directly test the impact of ecological pressures on viral spread during contact and the first adaptive steps of the virus during spillover can provide important insights. Specifically, the study of the emergence and adaption of hantaviruses can provide critical data for: (1) models for prediction of RNA viral emergence and mitigation; (2) identification of viral and host targets for therapeutic intervention; and (3) a basic understanding of the selective pressures that modulate viral-host interactions and viral genetic variation. Specific aims: We propose field and laboratory studies using South American hantaviruses as a model system to test specific selective pressures to reveal evolutionary mechanisms of RNA virus adaption in reservoir (HR) and spillover host (HS) host systems. The three Aims proposed will: (1) define viral emergence within host reservoirs and sympatric rodent species during extrinsic (environmental) pressure; (2) reveal the relationship of extrinsic (environment & ecological) pressures on preexisting intrahost genetic variation in wild rodent populations; and, (3) define viral fitness, virulence, and genotype during adaptation to new host species in vitro. Research design and methods: The proposed field site for Aim 1 lies in eastern Paraguay, where two hantaviruses co-circulate, and where elevated seroprevalence levels of hantaviruses are associated with anthropogenic alteration of land use. We propose to assess effects of: (1) increased food resources and (2) reduced predation on virus prevalence (Aim 1) and intra- and interhost genetic variation in rodents within native and disturbed forest areas (Aim 2). In Aim 3, we focus on the fitness and virulence of these viruses and the genetic changes that lead to adaptation. Using primary endothelial cell culture-based approaches, we will define the impact of intrinsic (host) selective pressures occurring when a new host species, HS, interacts with virus as it replicates, adapts to a new host host.